Neurochem Res DOI 10.1007/s11064-014-1473-1
OVERVIEW
The Glutamine–Glutamate/GABA Cycle: Function, Regional Differences in Glutamate and GABA Production and Effects of Interference with GABA Metabolism Anne B. Walls • Helle S. Waagepetersen • Lasse K. Bak • Arne Schousboe • Ursula Sonnewald
Received: 18 August 2014 / Revised: 30 October 2014 / Accepted: 31 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The operation of a glutamine–glutamate/ GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a wellknown concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [14C]acetate or [13C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [13C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role
Special Issue: In honor of Michael Norenberg. A. B. Walls H. S. Waagepetersen L. K. Bak A. Schousboe Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark U. Sonnewald (&) Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Olav Kyrresgt. 3, 7489 Trondheim, Norway e-mail: [email protected]
in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake. Keywords Anaplerosis Glutamine Astrocytes Neurons Glutamate GABA Abbreviations GAD Glutamate decarboxylase GLN Glutamine GLU Glutamate GS Glutamine synthetase GVG c-VinylGABA KG a-Ketoglutarate KO Knockout MRS Magnetic resonance spectroscopy PC Pyruvate carboxylase PDH Pyruvate dehydrogenase TCA Tricarboxylic acid WT Wild type
The Glutamine–Glutamate/GABA Cycle The concept of compartmentation of metabolism in the brain and the existence of separate pools of metabolites in neurons and astrocytes is based on the observation that the specific radioactivity of glutamine could exceed that of its precursor glutamate, using certain radiolabelled substrates [1–4]. Thus, the concept of exchange of metabolites between compartments or cells was developed [5, 6]. Neurotransmitter glutamate is following release from the presynaptic neuron and interaction with receptors in the postsynaptic membrane removed from the synapse predominantly by sodium-coupled transport into astrocytes (Fig. 1) [7, 8]. This renders a need for replenishment of the neuronal transmitter pool of glutamate in
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Fig. 1 Schematic representation of key metabolic processes, release and uptake of neurotransmitters in glutamatergic and GABAergic synapses interacting with an astrocyte. The glutamate-glutamine cycle between the glutamatergic neuron and the astrocyte, and, analogously, the GABA/glutamate-glutamine cycle between the GABAergic neuron and the astrocyte, is shown. In the astrocytic compartment
pyruvate carboxylation to oxaloacetate via pyruvate carboxylase (PC) is indicated. One arrow can signify several reactions. GAD glutamate decarboxylase, GLN glutamine, GLU glutamate, GS glutamine synthetase, KG a-ketoglutarate, PC pyruvate carboxylase, PDH pyruvate dehydrogenase, TCA tricarboxylic acid
order to maintain neurotransmitter homeostasis. Hence, after uptake into astrocytes, glutamate may be converted to glutamine via glutamine synthetase which is exclusively expressed in glial cells [9]. Glutamine can subsequently be transferred from astrocytes to neurons and re-converted to glutamate. Altogether these reactions constitute the glutamine–glutamate cycle which is outlined in Fig. 1. Glutamine released by astrocytes can also function as precursor for the inhibitory neurotransmitter c-aminobutyric acid (GABA) via glutamate [10, 11] extending the concept to a glutamine–glutamate– GABA cycle (Fig. 1). It should be noted that GABA is removed from the extrasynaptic space predominantly by transporters in the presynaptic neurons and to a lesser extent into astrocytes [12]. In addition it has been demonstrated that GABA taken up by the pre-synaptic neuron contributes predominantly to the vesicular GABA pool in these neurons while a smaller fraction enters the metabolic pool [13]. Therefore, the loss of carbon skeletons from GABAergic neurons to astrocytes is less pronounced than that from glutamatergic neurons and it may be assumed that glutamine transfer from astrocytes to GABAergic neurons is less important than to glutamatergic neurons.
It should be noted that for net synthesis of compounds like glutamate, GABA and glutamine ‘‘new’’ tricarboxylic acid (TCA) cycle intermediates have to enter the cycle. In the brain this is preferentially or exclusively achieved by the anaplerotic enzyme pyruvate carboxylase (PC) [14, 15], which has a glial localization [16–18]. Pyruvate carboxylation generates a ‘‘new’’ molecule of oxaloacetate, which may condense with acetyl CoA to provide, after several steps, net synthesis of the TCA cycle intermediate aketoglutarate, from which glutamate can be formed and subsequently GABA or glutamine [19]. In both astrocytes and neurons glutamate, glutamine and GABA may be degraded by metabolism in the TCA cycle in order to maintain cellular metabolite homeostasis. It should be mentioned that pyruvate recycling is required for full oxidation of glutamate, glutamine and GABA, and to the extent that this pathway is active, there is a requirement for synthesis of ‘‘new’’ TCA cycle intermediates to uphold cerebral amino acid (neurotransmitter) homeostasis [20]. The described metabolic pathways (Fig. 1) demonstrate the many possibilities for metabolism of neurotransmitter glutamate and GABA, and it should be emphasized that the
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Fig. 2 Labelling patterns of the tricarboxylic (TCA) cycle intermediate citrate as well as in the TCA cycle related amino acids glutamate, glutamine, aspartate and GABA originating from metabolism of [1,2-13C]acetate generated from the first and subsequent turns of the cycle. Unlabelled oxaloacetate condenses with [1,2-13C] acetyl CoA to form 13C labelled citrate, isocitrate and a-ketoglutarate
the latter of which can be converted to [4,5-13C]glutamate and subsequently [4,5-13C]glutamine in astrocytes or [1,2-13C]GABA in neurons. Full or striped circles represent 13C (grey from 1st turn, striped from second turn and black from 3rd turn with [1,2-13C]acetyl CoA) and empty circles 12C (not labelled)
carbon skeleton taken up by astrocytes in the form of glutamate or GABA is not always the same as that returned to the neuron in the form of glutamine. However, the amount of glutamine transferred to the neurons must equal that of glutamate or GABA taken up by astrocytes plus the amount oxidized in the neurons in order to uphold neurotransmitter homeostasis. For more extensive, recent reviews the reader is referred to [12, 21, 22].
metabolism. Originally, [14C]acetate was used to establish the basis of the glutamine–glutamate/GABA cycle [3]. Later [13C]acetate in combination with magnetic resonance spectroscopy (MRS) or mass spectrometry has been utilized to obtain more detailed information about astrocytic metabolism. Acetate is particularly useful since it has been known for several decades that it is metabolized in astrocytes but not neurons [5, 11, 23, 24]. It was suggested that this is due to the abundance of special transport systems with a much higher expression level in astrocytes compared to neurons [25]. Acetate is accumulated by brain cortical tissue slices to concentrations in excess of those in the media, suggesting transporter mediated uptake, possibly via the sodium dependent monocarboxylate transporter [26]. It was shown that acetate is a good substrate for the neuronal monocarboxylate transporter MCT2 and the glial
Tools to Study the Glutamine–Glutamate/GABA Cycle Acetate Can be Used to Map Astrocyte Metabolism Isotopically labelled acetate has been used extensively as an important tool for the characterization of astrocyte
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MCT1 but a poor substrate for the glial MCT4, indicating that transport is not the distinguishing factor [26]. In order to interpret the data obtained from ex vivo or in vitro experiments with [13C]acetate it is necessary to have knowledge about the labelling patterns generated by the different metabolic pathways. This labelling is demonstrated using [1,2-13C]acetate as an example (Fig. 2). After uptake into the astrocytes [1,2-13C]acetate is converted to [1,2-13C]acetyl CoA which enters the TCA cycle and labels a-[4,5-13C]ketoglutarate and subsequently [4,5-13C]glutamate, [4,5-13C]glutamine and [1,2-13C]GABA. In subsequent turns of the TCA cycle the label will be distributed throughout the molecules as indicated in Fig. 2.
Glutamine Metabolism Studied in Brain Cell Cultures and In Vivo
note that fluorocitrate did not decrease the amount of GABA after intracerebral microinjection in vivo in mice but only that of glutamate and aspartate [27]. Cultured astrocytes exhibited a decreased formation of lactate in the presence of fluorocitrate [26], indicating that astrocytes to a considerable extent synthesize pyruvate and hence lactate from TCA cycle intermediates as discussed in detail in a recent review [20]. Glial-neuronal interchange of amino acids was also studied using [1,2-13C]acetate in fluoroacetate-treated mice [31]. Like fluorocitrate, fluoroacetate is an inhibitor of aconitase [29, 30]. Fluoroacetate blocked the glial, but not the neuronal TCA cycle activity as seen from the 13C labelling of glutamine, glutamate, and GABA [32].
The Glutamine–Glutamate/GABA Cycle Studied by Ex Vivo 13C MRS
Blocking Glutamine Synthesis Using cell cultures of cortical astrocytes, neurons and cocultures thereof it was possible to show that glutamine from astrocytes labels GABA in neurons [11]. The above mentioned cell cultures were incubated with medium containing [2-13C]acetate in the presence or absence of the GS inhibitor methionine sulfoximine (MSO). There was no significant metabolism of [2-13C]acetate in neurons but in astrocytes 13C labelling was observed in metabolites such as glutamate and glutamine. Using [2-13C]acetate as substrate the co-cultures envisaged astrocyte metabolism but also GABA was 13C labelled by neuronal metabolism. Labelling of glutamine was abolished in the presence of MSO and so was that of GABA showing that glutamine from astrocytes was the direct precursor of GABA in neurons. Analogous experiments performed in vivo in mice similarly showed that both 13C labelling and the amount of GABA was reduced in the presence of MSO which was applied via intracerebral microinjection [27] clearly indicating that glutamine is of quantitative importance for GABA synthesis in GABAergic neurons. Surprisingly, there was no decrease in the amount of labelling of glutamate from [2-13C]acetate after intracerebral microinjection of MSO [27]. Fluorocitrate and Fluoroacetate Another astrocyte preferring inhibitor is fluorocitrate which blocks the TCA cycle enzyme aconitase. It has been shown to inhibit the TCA cycle in astrocytes almost completely and, as expected, had little effect on cerebellar neurons [28]. Glutamine production, which takes place downstream of the TCA cycle was found to be significantly decreased in the presence of fluorocitrate [29, 30]. It is interesting to
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As mentioned above, [1,2-13C]acetate is metabolized predominantly by astrocytes, and gives rise to [4,5-13C]glutamate formation in astrocytes after metabolism in the TCA cycle. [4,5-13C]Glutamate is precursor for [4,5-13C]glutamine which is subsequently transferred from the astrocytes to neurons and reconverted to [4,5-13C]glutamate. However, since the amount of glutamate located in glutamatergic neurons accounts for over 80 % of the total glutamate pool [33, 34], [4,5-13C]glutamate quantified by 13C MRS predominantly reflects neuronal conversion of [4,5-13C]glutamine to [4,5-13C]glutamate. This amount will depend on the percent 13C enrichment of glutamine with [4,5-13C]glutamine. Information about transfer of glutamine from astrocytes to glutamatergic neurons can be obtained by dividing the amount of [4,5-13C]glutamate by the percent enrichment of glutamine with [4,5-13C]glutamine. The transfer of [4,5-13C]glutamine from astrocytes to GABAergic neurons can be estimated by the amount of [1,2-13C]GABA divided by the percent enrichment of glutamine with [4,5-13C]glutamine. Since GABA labelling and amount are relatively small it was only possible to quantify reliably when only [1,2-13C]acetate was injected without co-injection of [1-13C] glucose. The numbers provided in Table 1 give information about the amounts of glutamine transferred to glutamatergic neurons in cortex, cerebellum and hippocampus and in Table 2 to glutamatergic and GABAergic neurons in cortex during the time of the experiment, i.e. from injection of the 13 C labelled precursors to euthanasia of the animals. The numbers presented in Tables 1 and 2 are not absolute since the experiments were not carried out under steady state conditions. They are under-estimations since these values are not corrected for the amount of [4,5-13C]glutamine being oxidatively metabolized in the neuron. Furthermore, the amounts of [4,5-13C]glutamine metabolized in the neurons
Neurochem Res Table 1 Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic neurons in cortex, cerebellum and hippocampus from mice injected with [1,2-13C]acetate in combination with [1-13C]glucose Cortex
Cerebellum
Hippocampus
WTC57BL/6
36.6 ± 1.1
nq
48.5 ± 6.2
GAD65 KO
35.9 ± 2.5
nq
43.9 ± 3.4
WTFVB/N
17.7 ± 1.4
13.6 ± 0.9#
nq
TG73.7
18.8 ± 1.4
14.1 ± 1.5
nq
Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic neurons in two mouse strains and gene-modified versions of these. Glutamate decarboxylase (GAD) 65 knockout (KO) mice were backcrossed for 10 generations to a C57BL/6 background and littermate GAD65 ?/? mice (WTC57BL/6) were used as control [34]. Transgenic mice (TG) from the mouse line Tg73.7 overexpressing human glial fibrillary acidic protein (GFAP) which produces astrocytic aggregates were generated on a FVB/N background and compared to controls (WTFVB/N) [35]. The mice were injected with a combination of [1,2-13C]acetate and [1-13C]glucose and sacrificed 15 min later as detailed in the publications [34, 35]. The presented data were calculated by dividing the amounts of [4,5-13C] glutamate labelled from [1,2-13C]acetate by the enrichment (%) of glutamine with [4,5-13C]glutamine labelled from [1,2-13C]acetate. The numbers presented are not absolute since the experiments were not carried out under steady state conditions. They can, however, be used to compare glutamine transfer in different brain regions and among GABAergic and glutamatergic neurons. Results are averages ± SEM, n = 3–8; Student’s t test was used for statistical analysis GAD glutamate decarboxylase, KO knock out, nq not quantified, TG transgenic, WT wild type * Different from group above,
#
Table 2 Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic and GABAergic neurons in cortex from mice injected with [1,2-13C]acetate Cortex Glutamatergic
GABAergic
WTC57BL/6
24.0 ± 1.4
3.0 ± 0.3§
GAD65 KO
20.7 ± 2.9
1.6 ± 0.1*§
WTC57BL/6?GVG
21.0 ± 1.4
3.2 ± 0.5§
GAD65 KO?GVG
18.5 ± 2.5
1.6 ± 0.4§
Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic and GABAergic neurons, respectively. Glutamate decarboxylase (GAD) 65 knockout (KO) mice were backcrossed for 10 generations to a C57BL/6 background and littermate GAD65 ?/? mice (WTC57BL/6) served as control. The mice were injected with NaCl or GVG and were 24 h later injected with [1,2-13C]acetate and sacrificed 15 min later as detailed in the publication [34]. The presented data were calculated by dividing the amounts of [4,5-13C] glutamate and [1,2-13C]GABA labelled from [1,2-13C]acetate by the % enrichment of glutamine with [4,5-13C]glutamine labelled from [1,2-13C]acetate. The numbers presented are not absolute since the experiments were not carried out under steady state conditions. They can, however, be used to compare glutamine transfer in different brain regions and among GABAergic and glutamatergic neurons. Results are averages ± SEM, n = 5–8; Student’s t test was used for statistical analysis GAD glutamate decarboxylase, GVG c-vinyl GABA, KO knock out, WT wild type * Different from group above, tergic neurons
§
different from transfer to glutama-
different from cortex
are not known. In spite of this they can be used to compare glutamine transfer in different brain regions and GABAergic and among glutamatergic neurons. Amount of Glutamine Transferred to Glutamatergic Neurons in Cortex, Cerebellum and Hippocampus Wild type FVB/N (WTFVB/N) and C57BL/6 (WTC57BL/6) mice were injected with a combination of [1,2-13C]acetate and [1-13C]glucose [35, 36] and detailed mapping of both astrocytic and neuronal metabolism was obtained by MRS analysis of tissue extracts of different brain areas. In cerebral cortex almost twice as much glutamine was transferred to glutamatergic neurons in WTC57BL/6 mice compared to in WTFVB/N mice underlining the existence of inter-strain differences in mice. The amount of glutamine transported to the glutamatergic neurons in the cerebellum was smaller compared to the cortex of the WTFVB/N mice. In (WTC57BL/6) mice there was a tendency of more glutamine being transported to the glutamatergic neurons in hippocampus compared to that transferred to cortex of these mice, but this was not statistically significant. It was
unfortunately not possible to reliably quantify the transport of glutamine to the GABAergic neurons in mice when [1-13C]glucose was co-injected with the [1,2-13C]acetate.
Amount of Glutamine Transferred to Glutamatergic and GABAergic Neurons in Cortex and to Glutamatergic Neurons in Hippocampus in GAD65 Knockout Animals GABA synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) which has been identified in two isoforms, i.e. GAD65 and GAD67 (references in [35]) of which GAD65 has repeatedly been shown to be important during intensified synaptic activity [37, 38]. In order to specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intra- and inter-cellular GABA homeostasis, GAD65 knockout (KO) and corresponding wild type mice (WTC57BL/6) were injected with [1,2-13C]acetate and [1-13C]glucose [35]. It was revealed that GAD65 is crucial for the maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate [34]. GAD67 was found to be important for synthesis of GABA
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from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism [35]. Moreover, it was demonstrated that the total amount of GABA was reduced by approximately 20 % in mice lacking GAD65 [34]. In contrast, the amount of glutamate and the 13C labelling of glutamate from [1,2-13C]acetate were unaltered in GAD65 KO mice compared to WTC57BL/6. This is supported by the finding that there was no difference in the transfer of glutamine to glutamatergic neurons between wild type and GAD65 KO mice in neither cortex nor hippocampus. Synthesis of GABA from glutamine in the GABAergic synapses was investigated in GAD65 knockout and wild type mice using [1,2-13C]acetate and in some cases c-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation [35, 39]. Also when injecting [1,2-13C]acetate without co-injection of [1-13C]glucose, we find that glutamine transfer to glutamatergic neurons is maintained in GAD65 KO mice. However, glutamine transfer to GABAergic neurons (Table 2) was significantly lower in the GAD65 KO mice compared to controls and this likely to be related to the lower GABA content observed in these mice [35]. GVG treatment led to an increase in GABA content in cerebral cortex which was approximately three times higher than that in mice not treated with GVG, and this effect was independent of genotype [34]. It should be noted though that the reduction in GABA content of approximately 20 % in the GAD65 knockout animals compared to controls was preserved following GVG treatment [34]. Glutamine transfer to GABAergic neurons was, however, not affected by GVG neither in control nor in GAD65 KO mice (Table 2), indicating that glutamine transfer and GABA biosynthesis operate independent of GABA degradation. In cortex of both genotypes considerably more glutamine was transported to glutamatergic than GABAergic neurons (Table 2). Considering the fact that glutamate is removed from the synapse via glial uptake whereas GABA is reentering GABAergic neurons [40] and that excitatory neurons outnumber inhibitory cells by a factor of 9–1, and 90 % of synapses release glutamate [41] the amount of glutamine going into GABAergic neurons seems high compared to that going into glutamatergic neurons. It can be concluded that glutamine also has an important role in GABAergic neurons compatible with the finding that the cerebral GABA content was reduced in mice receiving intracerebral injections of MSO, as discussed above [27]. In an earlier in vivo MRS study it was shown that the GABA/glutamateglutamine cycle flux comprised 23 % of total (glutamate plus GABA) neurotransmitter cycling in halothane anesthetised rats [42]. It should be pointed out though that halothane anesthesia reduces glutamine flux to glutamatergic neurons [43].
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Amount of Glutamine Transferred to Glutamatergic Neurons in Cortex and Cerebellum of TG73.7 Animals Alexander disease is a rare and usually fatal neurological disorder resulting from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP) and is characterized by the abundant presence of protein aggregates in astrocytes [43]. A transgenic mouse line (Tg73.7) over-expressing human GFAP produces astrocytic aggregates, which are indistinguishable from those seen in humans suffering from Alexander disease [43]. To investigate possible metabolic changes associated with Alexander disease, Tg73.7 mice and controls (WTFVB/N) were injected with a combination of [1,2-13C]acetate and [1-13C]glucose [36]. Brain extracts were analysed by MRS and in the cerebral cortex, reduced utilization of [1,2-13C]acetate for synthesis of glutamine, glutamate, and GABA was observed [35]. This was assumed to indicate decreased transfer of glutamine from astrocytes to neurons compared with control mice. However, when calculating the transfer of glutamine to glutamatergic neurons in the above mentioned way, no differences were found in cerebral cortex or cerebellum (Table 1). This indicates that the lower amounts of [4,5-13C]glutamate labelled from [1,2-13C]acetate is solely the consequence of a reduction in the extent of their precursor [4,5-13C]glutamine being labelled from [1,2-13C]acetate and not from a reduced transfer of glutamine to neurons. Accordingly, transfer of glutamine to the neuronal compartments seems to be maintained even when astrocytic metabolism is compromised.
In Conclusion Even though it is thought that most neurotransmitter glutamate is taken up into astrocytes and has to be returned to the glutamatergic neurons in the form of glutamine whereas GABAergic neurons recycle GABA and furthermore approximately 90 % of neurons in cortex are glutamatergic, only approximately 80 % of glutamine transferred from astrocytes to neurons is transported into glutamatergic neurons. This shows that glutamine also has an important role in GABAergic neurons. Acknowledgments The writing of this review was supported by a Grant to Ph.D. Anne B. Walls by the Danish Medical Research Council [Grant number 0602-01660B].
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GJ, Albers RW, Brady ST, Price DL (eds) Basic neurochemistry, 8th edn. Elsevier Inc, London, pp 200–231 Schousboe A (2012) Studies of brain metabolism: a historical perspective. Advances in neurobiology, vol 4. Springer Science, NY, pp 909–920 Minchin MC, Beart PM (1975) Compartmentation of amino acid metabolism in the rat posterior pituitary. J Neurochem 24(5):881–884 Muir D, Berl S, Clarke DD (1986) Acetate and fluoroacetate as possible markers for glial metabolism in vivo. Brain Res 380(2):336–340 Waniewski RA, Martin DL (1998) Preferential utilization of acetate by astrocytes is attributable to transport. J Neurosci 18(14):5225–5233 Rae C, Fekete AD, Kashem MA, Nasrallah FA, Broer S (2012) Metabolism, compartmentation, transport and production of acetate in the cortical brain tissue slice. Neurochem Res 37(11):2541–2553. doi:10.1007/s11064-012-0847-5 Fonnum F, Johnsen A, Hassel B (1997) Use of fluorocitrate and fluoroacetate in the study of brain metabolism. Glia 21(1):106–113. doi:10.1002/(SICI)1098-1136(199709)21:1\106:AID-GLIA12[ 3.0.CO;2-W Hassel B, Westergaard N, Schousboe A, Fonnum F (1995) Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate. Neurochem Res 20(4):413–420 Clarke DD (1991) Fluoroacetate and fluorocitrate: mechanism of action. Neurochem Res 16(9):1055–1058 Paulsen RE, Contestabile A, Villani L, Fonnum F (1987) An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate. J Neurochem 48(5):1377–1385 Hassel B, Bachelard H, Jones P, Fonnum F, Sonnewald U (1997) Trafficking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by fluoroacetate. J Cereb Blood Flow Metab 17(11):1230–1238 Hassel B, Bachelard H, Jones P, Fonnum F, Sonnewald U (1997) Trafficking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by fluoroacetate. J Cereb Blood Flow Metab 17(11):1230–1238. doi:10.1097/00004647199711000-00012 Ottersen OP, Storm-Mathisen J (1985) Different neuronal localization of aspartate-like and glutamate-like immunoreactivities in the hippocampus of rat, guinea-pig and Senegalese baboon (Papio papio), with a note on the distribution of gamma-aminobutyrate. Neuroscience 16(3):589–606 Ottersen OP, Zhang N, Walberg F (1992) Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46(3):519–534. doi:10.1016/0306-4522(92)90141-N Walls AB, Eyjolfsson EM, Smeland OB, Nilsen LH, Schousboe I, Schousboe A, Sonnewald U, Waagepetersen HS (2010) Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine. J Cereb Blood Flow Metab 31(2):494–503. doi:10.1038/jcbfm.2010.115 Meisingset TW, Risa O, Brenner M, Messing A, Sonnewald U (2010) Alteration of glial-neuronal metabolic interactions in a mouse model of Alexander disease. Glia 58(10):1228–1234. doi:10.1002/glia.21003 Tian N, Petersen C, Kash S, Baekkeskov S, Copenhagen D, Nicoll R (1999) The role of the synthetic enzyme GAD65 in the control of neuronal gamma-aminobutyric acid release. Proc Natl Acad Sci U S A 96(22):12911–12916 Choi SY, Morales B, Lee HK, Kirkwood A (2002) Absence of long-term depression in the visual cortex of glutamic acid decarboxylase-65 knock-out mice. J Neurosci 22(13):5271–5276 Rowley NM, Madsen KK, Schousboe A, Steve White H (2012) Glutamate and GABA synthesis, release, transport and
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OVERVIEW
The Glutamine–Glutamate/GABA Cycle: Function, Regional Differences in Glutamate and GABA Production and Effects of Interference with GABA Metabolism Anne B. Walls • Helle S. Waagepetersen • Lasse K. Bak • Arne Schousboe • Ursula Sonnewald
Received: 18 August 2014 / Revised: 30 October 2014 / Accepted: 31 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The operation of a glutamine–glutamate/ GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a wellknown concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [14C]acetate or [13C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [13C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role
Special Issue: In honor of Michael Norenberg. A. B. Walls H. S. Waagepetersen L. K. Bak A. Schousboe Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark U. Sonnewald (&) Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Olav Kyrresgt. 3, 7489 Trondheim, Norway e-mail: [email protected]
in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake. Keywords Anaplerosis Glutamine Astrocytes Neurons Glutamate GABA Abbreviations GAD Glutamate decarboxylase GLN Glutamine GLU Glutamate GS Glutamine synthetase GVG c-VinylGABA KG a-Ketoglutarate KO Knockout MRS Magnetic resonance spectroscopy PC Pyruvate carboxylase PDH Pyruvate dehydrogenase TCA Tricarboxylic acid WT Wild type
The Glutamine–Glutamate/GABA Cycle The concept of compartmentation of metabolism in the brain and the existence of separate pools of metabolites in neurons and astrocytes is based on the observation that the specific radioactivity of glutamine could exceed that of its precursor glutamate, using certain radiolabelled substrates [1–4]. Thus, the concept of exchange of metabolites between compartments or cells was developed [5, 6]. Neurotransmitter glutamate is following release from the presynaptic neuron and interaction with receptors in the postsynaptic membrane removed from the synapse predominantly by sodium-coupled transport into astrocytes (Fig. 1) [7, 8]. This renders a need for replenishment of the neuronal transmitter pool of glutamate in
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Fig. 1 Schematic representation of key metabolic processes, release and uptake of neurotransmitters in glutamatergic and GABAergic synapses interacting with an astrocyte. The glutamate-glutamine cycle between the glutamatergic neuron and the astrocyte, and, analogously, the GABA/glutamate-glutamine cycle between the GABAergic neuron and the astrocyte, is shown. In the astrocytic compartment
pyruvate carboxylation to oxaloacetate via pyruvate carboxylase (PC) is indicated. One arrow can signify several reactions. GAD glutamate decarboxylase, GLN glutamine, GLU glutamate, GS glutamine synthetase, KG a-ketoglutarate, PC pyruvate carboxylase, PDH pyruvate dehydrogenase, TCA tricarboxylic acid
order to maintain neurotransmitter homeostasis. Hence, after uptake into astrocytes, glutamate may be converted to glutamine via glutamine synthetase which is exclusively expressed in glial cells [9]. Glutamine can subsequently be transferred from astrocytes to neurons and re-converted to glutamate. Altogether these reactions constitute the glutamine–glutamate cycle which is outlined in Fig. 1. Glutamine released by astrocytes can also function as precursor for the inhibitory neurotransmitter c-aminobutyric acid (GABA) via glutamate [10, 11] extending the concept to a glutamine–glutamate– GABA cycle (Fig. 1). It should be noted that GABA is removed from the extrasynaptic space predominantly by transporters in the presynaptic neurons and to a lesser extent into astrocytes [12]. In addition it has been demonstrated that GABA taken up by the pre-synaptic neuron contributes predominantly to the vesicular GABA pool in these neurons while a smaller fraction enters the metabolic pool [13]. Therefore, the loss of carbon skeletons from GABAergic neurons to astrocytes is less pronounced than that from glutamatergic neurons and it may be assumed that glutamine transfer from astrocytes to GABAergic neurons is less important than to glutamatergic neurons.
It should be noted that for net synthesis of compounds like glutamate, GABA and glutamine ‘‘new’’ tricarboxylic acid (TCA) cycle intermediates have to enter the cycle. In the brain this is preferentially or exclusively achieved by the anaplerotic enzyme pyruvate carboxylase (PC) [14, 15], which has a glial localization [16–18]. Pyruvate carboxylation generates a ‘‘new’’ molecule of oxaloacetate, which may condense with acetyl CoA to provide, after several steps, net synthesis of the TCA cycle intermediate aketoglutarate, from which glutamate can be formed and subsequently GABA or glutamine [19]. In both astrocytes and neurons glutamate, glutamine and GABA may be degraded by metabolism in the TCA cycle in order to maintain cellular metabolite homeostasis. It should be mentioned that pyruvate recycling is required for full oxidation of glutamate, glutamine and GABA, and to the extent that this pathway is active, there is a requirement for synthesis of ‘‘new’’ TCA cycle intermediates to uphold cerebral amino acid (neurotransmitter) homeostasis [20]. The described metabolic pathways (Fig. 1) demonstrate the many possibilities for metabolism of neurotransmitter glutamate and GABA, and it should be emphasized that the
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Fig. 2 Labelling patterns of the tricarboxylic (TCA) cycle intermediate citrate as well as in the TCA cycle related amino acids glutamate, glutamine, aspartate and GABA originating from metabolism of [1,2-13C]acetate generated from the first and subsequent turns of the cycle. Unlabelled oxaloacetate condenses with [1,2-13C] acetyl CoA to form 13C labelled citrate, isocitrate and a-ketoglutarate
the latter of which can be converted to [4,5-13C]glutamate and subsequently [4,5-13C]glutamine in astrocytes or [1,2-13C]GABA in neurons. Full or striped circles represent 13C (grey from 1st turn, striped from second turn and black from 3rd turn with [1,2-13C]acetyl CoA) and empty circles 12C (not labelled)
carbon skeleton taken up by astrocytes in the form of glutamate or GABA is not always the same as that returned to the neuron in the form of glutamine. However, the amount of glutamine transferred to the neurons must equal that of glutamate or GABA taken up by astrocytes plus the amount oxidized in the neurons in order to uphold neurotransmitter homeostasis. For more extensive, recent reviews the reader is referred to [12, 21, 22].
metabolism. Originally, [14C]acetate was used to establish the basis of the glutamine–glutamate/GABA cycle [3]. Later [13C]acetate in combination with magnetic resonance spectroscopy (MRS) or mass spectrometry has been utilized to obtain more detailed information about astrocytic metabolism. Acetate is particularly useful since it has been known for several decades that it is metabolized in astrocytes but not neurons [5, 11, 23, 24]. It was suggested that this is due to the abundance of special transport systems with a much higher expression level in astrocytes compared to neurons [25]. Acetate is accumulated by brain cortical tissue slices to concentrations in excess of those in the media, suggesting transporter mediated uptake, possibly via the sodium dependent monocarboxylate transporter [26]. It was shown that acetate is a good substrate for the neuronal monocarboxylate transporter MCT2 and the glial
Tools to Study the Glutamine–Glutamate/GABA Cycle Acetate Can be Used to Map Astrocyte Metabolism Isotopically labelled acetate has been used extensively as an important tool for the characterization of astrocyte
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MCT1 but a poor substrate for the glial MCT4, indicating that transport is not the distinguishing factor [26]. In order to interpret the data obtained from ex vivo or in vitro experiments with [13C]acetate it is necessary to have knowledge about the labelling patterns generated by the different metabolic pathways. This labelling is demonstrated using [1,2-13C]acetate as an example (Fig. 2). After uptake into the astrocytes [1,2-13C]acetate is converted to [1,2-13C]acetyl CoA which enters the TCA cycle and labels a-[4,5-13C]ketoglutarate and subsequently [4,5-13C]glutamate, [4,5-13C]glutamine and [1,2-13C]GABA. In subsequent turns of the TCA cycle the label will be distributed throughout the molecules as indicated in Fig. 2.
Glutamine Metabolism Studied in Brain Cell Cultures and In Vivo
note that fluorocitrate did not decrease the amount of GABA after intracerebral microinjection in vivo in mice but only that of glutamate and aspartate [27]. Cultured astrocytes exhibited a decreased formation of lactate in the presence of fluorocitrate [26], indicating that astrocytes to a considerable extent synthesize pyruvate and hence lactate from TCA cycle intermediates as discussed in detail in a recent review [20]. Glial-neuronal interchange of amino acids was also studied using [1,2-13C]acetate in fluoroacetate-treated mice [31]. Like fluorocitrate, fluoroacetate is an inhibitor of aconitase [29, 30]. Fluoroacetate blocked the glial, but not the neuronal TCA cycle activity as seen from the 13C labelling of glutamine, glutamate, and GABA [32].
The Glutamine–Glutamate/GABA Cycle Studied by Ex Vivo 13C MRS
Blocking Glutamine Synthesis Using cell cultures of cortical astrocytes, neurons and cocultures thereof it was possible to show that glutamine from astrocytes labels GABA in neurons [11]. The above mentioned cell cultures were incubated with medium containing [2-13C]acetate in the presence or absence of the GS inhibitor methionine sulfoximine (MSO). There was no significant metabolism of [2-13C]acetate in neurons but in astrocytes 13C labelling was observed in metabolites such as glutamate and glutamine. Using [2-13C]acetate as substrate the co-cultures envisaged astrocyte metabolism but also GABA was 13C labelled by neuronal metabolism. Labelling of glutamine was abolished in the presence of MSO and so was that of GABA showing that glutamine from astrocytes was the direct precursor of GABA in neurons. Analogous experiments performed in vivo in mice similarly showed that both 13C labelling and the amount of GABA was reduced in the presence of MSO which was applied via intracerebral microinjection [27] clearly indicating that glutamine is of quantitative importance for GABA synthesis in GABAergic neurons. Surprisingly, there was no decrease in the amount of labelling of glutamate from [2-13C]acetate after intracerebral microinjection of MSO [27]. Fluorocitrate and Fluoroacetate Another astrocyte preferring inhibitor is fluorocitrate which blocks the TCA cycle enzyme aconitase. It has been shown to inhibit the TCA cycle in astrocytes almost completely and, as expected, had little effect on cerebellar neurons [28]. Glutamine production, which takes place downstream of the TCA cycle was found to be significantly decreased in the presence of fluorocitrate [29, 30]. It is interesting to
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As mentioned above, [1,2-13C]acetate is metabolized predominantly by astrocytes, and gives rise to [4,5-13C]glutamate formation in astrocytes after metabolism in the TCA cycle. [4,5-13C]Glutamate is precursor for [4,5-13C]glutamine which is subsequently transferred from the astrocytes to neurons and reconverted to [4,5-13C]glutamate. However, since the amount of glutamate located in glutamatergic neurons accounts for over 80 % of the total glutamate pool [33, 34], [4,5-13C]glutamate quantified by 13C MRS predominantly reflects neuronal conversion of [4,5-13C]glutamine to [4,5-13C]glutamate. This amount will depend on the percent 13C enrichment of glutamine with [4,5-13C]glutamine. Information about transfer of glutamine from astrocytes to glutamatergic neurons can be obtained by dividing the amount of [4,5-13C]glutamate by the percent enrichment of glutamine with [4,5-13C]glutamine. The transfer of [4,5-13C]glutamine from astrocytes to GABAergic neurons can be estimated by the amount of [1,2-13C]GABA divided by the percent enrichment of glutamine with [4,5-13C]glutamine. Since GABA labelling and amount are relatively small it was only possible to quantify reliably when only [1,2-13C]acetate was injected without co-injection of [1-13C] glucose. The numbers provided in Table 1 give information about the amounts of glutamine transferred to glutamatergic neurons in cortex, cerebellum and hippocampus and in Table 2 to glutamatergic and GABAergic neurons in cortex during the time of the experiment, i.e. from injection of the 13 C labelled precursors to euthanasia of the animals. The numbers presented in Tables 1 and 2 are not absolute since the experiments were not carried out under steady state conditions. They are under-estimations since these values are not corrected for the amount of [4,5-13C]glutamine being oxidatively metabolized in the neuron. Furthermore, the amounts of [4,5-13C]glutamine metabolized in the neurons
Neurochem Res Table 1 Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic neurons in cortex, cerebellum and hippocampus from mice injected with [1,2-13C]acetate in combination with [1-13C]glucose Cortex
Cerebellum
Hippocampus
WTC57BL/6
36.6 ± 1.1
nq
48.5 ± 6.2
GAD65 KO
35.9 ± 2.5
nq
43.9 ± 3.4
WTFVB/N
17.7 ± 1.4
13.6 ± 0.9#
nq
TG73.7
18.8 ± 1.4
14.1 ± 1.5
nq
Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic neurons in two mouse strains and gene-modified versions of these. Glutamate decarboxylase (GAD) 65 knockout (KO) mice were backcrossed for 10 generations to a C57BL/6 background and littermate GAD65 ?/? mice (WTC57BL/6) were used as control [34]. Transgenic mice (TG) from the mouse line Tg73.7 overexpressing human glial fibrillary acidic protein (GFAP) which produces astrocytic aggregates were generated on a FVB/N background and compared to controls (WTFVB/N) [35]. The mice were injected with a combination of [1,2-13C]acetate and [1-13C]glucose and sacrificed 15 min later as detailed in the publications [34, 35]. The presented data were calculated by dividing the amounts of [4,5-13C] glutamate labelled from [1,2-13C]acetate by the enrichment (%) of glutamine with [4,5-13C]glutamine labelled from [1,2-13C]acetate. The numbers presented are not absolute since the experiments were not carried out under steady state conditions. They can, however, be used to compare glutamine transfer in different brain regions and among GABAergic and glutamatergic neurons. Results are averages ± SEM, n = 3–8; Student’s t test was used for statistical analysis GAD glutamate decarboxylase, KO knock out, nq not quantified, TG transgenic, WT wild type * Different from group above,
#
Table 2 Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic and GABAergic neurons in cortex from mice injected with [1,2-13C]acetate Cortex Glutamatergic
GABAergic
WTC57BL/6
24.0 ± 1.4
3.0 ± 0.3§
GAD65 KO
20.7 ± 2.9
1.6 ± 0.1*§
WTC57BL/6?GVG
21.0 ± 1.4
3.2 ± 0.5§
GAD65 KO?GVG
18.5 ± 2.5
1.6 ± 0.4§
Amounts of glutamine (nmol/g/15 min) transferred from astrocytes to glutamatergic and GABAergic neurons, respectively. Glutamate decarboxylase (GAD) 65 knockout (KO) mice were backcrossed for 10 generations to a C57BL/6 background and littermate GAD65 ?/? mice (WTC57BL/6) served as control. The mice were injected with NaCl or GVG and were 24 h later injected with [1,2-13C]acetate and sacrificed 15 min later as detailed in the publication [34]. The presented data were calculated by dividing the amounts of [4,5-13C] glutamate and [1,2-13C]GABA labelled from [1,2-13C]acetate by the % enrichment of glutamine with [4,5-13C]glutamine labelled from [1,2-13C]acetate. The numbers presented are not absolute since the experiments were not carried out under steady state conditions. They can, however, be used to compare glutamine transfer in different brain regions and among GABAergic and glutamatergic neurons. Results are averages ± SEM, n = 5–8; Student’s t test was used for statistical analysis GAD glutamate decarboxylase, GVG c-vinyl GABA, KO knock out, WT wild type * Different from group above, tergic neurons
§
different from transfer to glutama-
different from cortex
are not known. In spite of this they can be used to compare glutamine transfer in different brain regions and GABAergic and among glutamatergic neurons. Amount of Glutamine Transferred to Glutamatergic Neurons in Cortex, Cerebellum and Hippocampus Wild type FVB/N (WTFVB/N) and C57BL/6 (WTC57BL/6) mice were injected with a combination of [1,2-13C]acetate and [1-13C]glucose [35, 36] and detailed mapping of both astrocytic and neuronal metabolism was obtained by MRS analysis of tissue extracts of different brain areas. In cerebral cortex almost twice as much glutamine was transferred to glutamatergic neurons in WTC57BL/6 mice compared to in WTFVB/N mice underlining the existence of inter-strain differences in mice. The amount of glutamine transported to the glutamatergic neurons in the cerebellum was smaller compared to the cortex of the WTFVB/N mice. In (WTC57BL/6) mice there was a tendency of more glutamine being transported to the glutamatergic neurons in hippocampus compared to that transferred to cortex of these mice, but this was not statistically significant. It was
unfortunately not possible to reliably quantify the transport of glutamine to the GABAergic neurons in mice when [1-13C]glucose was co-injected with the [1,2-13C]acetate.
Amount of Glutamine Transferred to Glutamatergic and GABAergic Neurons in Cortex and to Glutamatergic Neurons in Hippocampus in GAD65 Knockout Animals GABA synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) which has been identified in two isoforms, i.e. GAD65 and GAD67 (references in [35]) of which GAD65 has repeatedly been shown to be important during intensified synaptic activity [37, 38]. In order to specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intra- and inter-cellular GABA homeostasis, GAD65 knockout (KO) and corresponding wild type mice (WTC57BL/6) were injected with [1,2-13C]acetate and [1-13C]glucose [35]. It was revealed that GAD65 is crucial for the maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate [34]. GAD67 was found to be important for synthesis of GABA
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from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism [35]. Moreover, it was demonstrated that the total amount of GABA was reduced by approximately 20 % in mice lacking GAD65 [34]. In contrast, the amount of glutamate and the 13C labelling of glutamate from [1,2-13C]acetate were unaltered in GAD65 KO mice compared to WTC57BL/6. This is supported by the finding that there was no difference in the transfer of glutamine to glutamatergic neurons between wild type and GAD65 KO mice in neither cortex nor hippocampus. Synthesis of GABA from glutamine in the GABAergic synapses was investigated in GAD65 knockout and wild type mice using [1,2-13C]acetate and in some cases c-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation [35, 39]. Also when injecting [1,2-13C]acetate without co-injection of [1-13C]glucose, we find that glutamine transfer to glutamatergic neurons is maintained in GAD65 KO mice. However, glutamine transfer to GABAergic neurons (Table 2) was significantly lower in the GAD65 KO mice compared to controls and this likely to be related to the lower GABA content observed in these mice [35]. GVG treatment led to an increase in GABA content in cerebral cortex which was approximately three times higher than that in mice not treated with GVG, and this effect was independent of genotype [34]. It should be noted though that the reduction in GABA content of approximately 20 % in the GAD65 knockout animals compared to controls was preserved following GVG treatment [34]. Glutamine transfer to GABAergic neurons was, however, not affected by GVG neither in control nor in GAD65 KO mice (Table 2), indicating that glutamine transfer and GABA biosynthesis operate independent of GABA degradation. In cortex of both genotypes considerably more glutamine was transported to glutamatergic than GABAergic neurons (Table 2). Considering the fact that glutamate is removed from the synapse via glial uptake whereas GABA is reentering GABAergic neurons [40] and that excitatory neurons outnumber inhibitory cells by a factor of 9–1, and 90 % of synapses release glutamate [41] the amount of glutamine going into GABAergic neurons seems high compared to that going into glutamatergic neurons. It can be concluded that glutamine also has an important role in GABAergic neurons compatible with the finding that the cerebral GABA content was reduced in mice receiving intracerebral injections of MSO, as discussed above [27]. In an earlier in vivo MRS study it was shown that the GABA/glutamateglutamine cycle flux comprised 23 % of total (glutamate plus GABA) neurotransmitter cycling in halothane anesthetised rats [42]. It should be pointed out though that halothane anesthesia reduces glutamine flux to glutamatergic neurons [43].
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Amount of Glutamine Transferred to Glutamatergic Neurons in Cortex and Cerebellum of TG73.7 Animals Alexander disease is a rare and usually fatal neurological disorder resulting from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP) and is characterized by the abundant presence of protein aggregates in astrocytes [43]. A transgenic mouse line (Tg73.7) over-expressing human GFAP produces astrocytic aggregates, which are indistinguishable from those seen in humans suffering from Alexander disease [43]. To investigate possible metabolic changes associated with Alexander disease, Tg73.7 mice and controls (WTFVB/N) were injected with a combination of [1,2-13C]acetate and [1-13C]glucose [36]. Brain extracts were analysed by MRS and in the cerebral cortex, reduced utilization of [1,2-13C]acetate for synthesis of glutamine, glutamate, and GABA was observed [35]. This was assumed to indicate decreased transfer of glutamine from astrocytes to neurons compared with control mice. However, when calculating the transfer of glutamine to glutamatergic neurons in the above mentioned way, no differences were found in cerebral cortex or cerebellum (Table 1). This indicates that the lower amounts of [4,5-13C]glutamate labelled from [1,2-13C]acetate is solely the consequence of a reduction in the extent of their precursor [4,5-13C]glutamine being labelled from [1,2-13C]acetate and not from a reduced transfer of glutamine to neurons. Accordingly, transfer of glutamine to the neuronal compartments seems to be maintained even when astrocytic metabolism is compromised.
In Conclusion Even though it is thought that most neurotransmitter glutamate is taken up into astrocytes and has to be returned to the glutamatergic neurons in the form of glutamine whereas GABAergic neurons recycle GABA and furthermore approximately 90 % of neurons in cortex are glutamatergic, only approximately 80 % of glutamine transferred from astrocytes to neurons is transported into glutamatergic neurons. This shows that glutamine also has an important role in GABAergic neurons. Acknowledgments The writing of this review was supported by a Grant to Ph.D. Anne B. Walls by the Danish Medical Research Council [Grant number 0602-01660B].
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